The recently discovered rhesus monkey protein TRIM5alpha (TRIM5alpha-rh) is responsible for the strong restriction imposed to human immunodeficiency virus (HIV-1) infection by these primates. We have correlated the restriction of HIV-1 by TRIM5alpha-rh with a decrease of viral core stability in the cytoplasm during infection. This suggested that TRIM5alpha-rh proteins block retroviral replication by changing the stability of the incoming retroviral core into the host cell. Separately, changes in the stability of the incoming retroviral core achieved by HIV-1 capsid mutagenesis results also in a loss of infectivity. Collectively, these findings suggested that modulation of viral core stability is detrimental for infection. Remarkably, we have found mutations in the B-box 2 domain of TRIM5alpha-rh that bind the HIV-1 incoming viral core, yet the stability or infectivity of the core is not affected. This particular set of B-box 2 domain mutants could bind capsid and oligomerize when compared to wild type. For these reasons, we hypothesize that binding to capsid by these mutants might be achieved differently than wild-type. In this proposal, we will test the hypothesis that cooperative binding of TRIM5alpha-rh trimers to the retroviral core results in core destabilization and inhibition of infection;and that cooperative binding of TRIM5alpha-rh trimers to the viral core is mediated by B-box 2-B-box 2 inter-trimer interactions. To investigate this hypothesis we will: 1) identify the B-box 2 determinants for retroviral restriction by structure-function studies;2) elucidate the role of B-box 2-B-box 2 interactions in retroviral restriction by TRIM5alpha-rh;and 3) assay cooperative binding of TRIM5alpha-rh trimers to the HIV-1 capsid. The completion of the proposed research will help me to achieve my long-term goals of becoming an independent investigator in the field of HIV-1/AIDS. For this reason the Dana-Farber Cancer Institute in combination with the laboratory of Dr. Joseph Sodroski is the adequate environment to pursue this goal. In addition, several training classes will be taken in order to smooth the transition from the mentored phase to the independent phase of the award. RELEVANCE (See instructions): The main goal of this proposal is to gain understanding on how the host factor TRIM5alpha potently blocks HIV-1 infection. In contrast to the rhesus monkey TRIM5alpha, the human homologue poorly restricts HIV-1. Understanding the anti-HIV-1 activity of TRIM5alpha rhesus will generate new therapeutic opportunities to improve the anti-HIV-1 activity of human TRIM5alpha.